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Northern Arabian Sea: Rare Fish Diversity and Biogeographic Affinities

PUJZ_39_2_191-212

Northern Arabian Sea: Rare Fish Diversity and Biogeographic Affinities

Bushra Sial1, Arif Muhammad Khan1, Mohsan Raza1,6*, Azhar Abbas Khan2, Sayyad Ali Raza Bukhari1, Muhammad Kashif Hanif3, Sher Khan Panhwar4, Muhammad Ashfaq5

1Department of Biotechnology, University of Sargodha

2Department of Entomology, University of Layyah

3Institute of Molecular Biology and Biotechnology, The University of Lahore, Sargodha Campus

4Centre of Excellence in Marine Biology, University of Karachi

5University of Guelph, Canada

6College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China

Abstract | Recent molecular approaches have revolutionized the world of species classification and identification. In this study, we delved into the fascinating domain of DNA barcoding precisely for rare marine species and delineated species population genetic variability, genetic differences, and phylogenetic relationships between families/genera. 542 COI sequences from experimental species and an online database were considered for phylogenetic and Fst analysis. Moreover, an online QR code generator was used to develop the first-ever QR codes for nucleotide information of these species. It is the first study from Pakistan to reveal the barcode gap, phylogenetic relationship, and genetic diversity of the fish species in the northern Arabian Sea. A notable genetic variation level was revealed, with the highest value of 0.75 indicating a significant differentiation between populations of Taiwan and Pakistan. In contrast, the lowest Fst value of 0.04 manifested minimal genetic differentiation between populations in the USA and Bangladesh. An average genetic distance using the Kimura 2 parameter (K2P) model using BOLD systems revealed 20.17 and 19.87 percent within genus and family respectively. Nevertheless, this study documented the COI sequence of Caesio varilineata and Uranoscopus dollfusi, for the first time. The combined use of taxonomy, DNA barcoding, and QR codes appeared to be robust approaches, and have paved the way for a better understanding of fishes rarely found in Pakistan, northern Arabian Sea.

Novelty Statement | This study demonstrates the utility of integrating DNA barcoding and QR coding for elucidating the genetic diversity and phylogenetic relationships of rare marine fish species in the northern Arabian Sea, thereby enhancing our understanding of this previously undercharacterized region.


Article History

Received: July 05, 2024

Revised: July 25, 2024

Accepted: August 07, 2024

Published: October 19, 2024

Authors’ Contributions

BS and MR performed research and wrote manuscript. AMK designed research, contributed reagents and analyzed data. AAK designed and performed research. SAB performed analysis. MKH did statistical/data analysis and proofreading. SKP performed research and MA analyzed sequence data.

Keywords

Northern Arabian sea, Genetic diversity, Phylogenetic analysis, Barcode gap, Genetic variations, Rare species

Copyright 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Corresponding author: Mohsan Raza

mohsanraxa124@gmail.com

To cite this article: Sial, B., Khan, A.M., Raza, M., Khan, A.A., Bukhari, S.A.R. Hanif, M.K., Panhwar, S.K., Ashfaq, M., 2024. Northern Arabian Sea: Rare fish diversity and biogeographic affinities. Punjab Univ. J. Zool., 39(2): 191-212. https://dx.doi.org/10.17582/journal.pujz/2024/39.2.191.212



Introduction

Due to contemporary advancements and the involvement of molecular tools, fish taxonomy has grabbed the interest of fisheries biologists globally. Pakistan is rich in fish diversity due to its subtropical position on the globe. Previously, conventional taxonomy was used as a major tool to describe species (Farooq and Panhwar, 2023; Qamar et al., 2016; Rauf et al., 2019). Conventional taxonomy based on just morphology has many limitations, as we are unable to identify processed meat, broken samples, or individuals at their early developmental stages (Keskin and Atar, 2013; Zhang and Hanner, 2012). Cryptic species, species having incomplete morphological characters, and novel species may not be identified accurately, leading to misidentification. Moreover, there is a knowledge gap for taxonomists to properly identify some fish groups at the species level. The classification description of fish in different literature is different which can also lead to misidentification by beginners. Currently, DNA barcoding is being successively used and has been proven as an efficient tool for the rapid and accurate identification of fish (DeSalle and Goldstein, 2019). Furthermore, the identification of cryptic species, species at earlier stages of their life, and processed samples having incomplete morphology can be more reliably executed using molecular techniques as compared to identification based on morphology (Galal-Khallaf et al., 2014; Raharinaivo et al., 2020; Wang et al., 2020). Molecular approaches together with morphological identification can serve as a potential tool in fish biology because they provide rapid, precise, and reasonable systems for identification. Moreover, the delineation of genetic distances within different species and even in the population of the same species can be measured using single gene-based studies (de Sousa et al., 2022; Habib et al., 2022; Khan et al., 2023; Tang et al., 2023). The 650 bp region of COI a mitochondrial gene is being extensively used in molecular identification (Lohman et al., 2009). This gene possesses great importance as it has a slow amino acid change rate (Hebert et al., 2003; Lynch and Jarrell, 1993). Practically, DNA barcoding using the COI gene is being worldwide used for the identification of freshwater and marine fish species with a success rate of up to 93% (Ward, 2012). However, there are some limitations of the COI gene too, such as partial lineage sorting phenomena and gene introgression, which may lead sometime towards misidentification (Eberle et al., 2020; Galimberti et al., 2021). So, by employing the combination of morphological and molecular techniques, many ambiguities can be removed. Moreover, many analysis tools in addition to the conventional morphological-based identification and DNA barcoding studies are applied to improve the accuracy of fish identification, which will further empower the discovery of cryptic species and will enrich the genetic diversity of fish species (Breman et al., 2016; Hou et al., 2018). Northern Arabian Sea possesses a complex and high ratio of biodiversity including abundantly found species as well as some unique and rarely occurring species. Normally the focus of researchers is on abundantly present fishes as they contribute a major part in daily fish landing. However, the study of rare fish holds significant importance for conservation, ecological, and scientific reasons. In this study, some rarely occurring fishes were tried to cover taxonomy at the molecular level. In total, 15 species belonging to 13 families and eight different classes were collected from daily fish landing facilities along the Karachi coast. By considering a 650 bp region of COI, a comprehensive DNA barcode database of 45 samples representing 15 marine species, 13 families, and 7 orders (Lactarius lactarius, Rachycentron canadum, Caesio varilineata, pempheris russellii, Pomacanthus annularis, Myripristis botche, Sargocentron rubrum, Plotosus lineatus, Chanos chanos, Uranoscopus dollfusi, Terapon jarbua, Terapon puta, Drepane longimana, Scatophagus argus, Pampus argenteus) was developed as these are rarely present in daily fish landing at Karachi coast.

This study aimed to provide robust taxonomic and molecular descriptions of rare fishes and to delineate the genetic variability, species population differentiation, and biogeographic affinities, to facilitate researchers for further findings on population structure, understanding of evolutionary processes, and conservation strategies for species inhabiting circumglobally.

Materials and Methods

Sampling and morphological identification

In total 542 sequences were used for analysis of genetic diversity in rarely occurring marine species. Detailed information on sampling and Genbank accession numbers of extracted sequences from each species is mentioned in Supplementary Table 1. Type species (Lactarius lactarius, Rachycentron canadum, Caesio varilineata, Pempheris russellii, Pomacanthus annularis, Myripristis botche, Sargocentron rubrum, Plotosus lineatus, Chanos chanos, Uranoscopus dollfusi, Terapon jarbua, Terapon puta, Drepane longimana, Scatophagus argus, Pampus argenteus) belonging to 13 families and 7 orders were collected from the commercial catches at the daily fish landing facility (Karachi Fisheries Harbor). The exact location of the sample collection is shown in Figure 1. Samples were transported to the fisheries laboratory, Centre of Excellence in Marine Biology (CEMB). Each species was identified based on its morphology by using taxonomic keys and an FAO field guide (Psomadakis, 2015). Photographs for each of the specimens were taken. Samples were then transported to the Department of Biotechnology, University of Sargodha for molecular analysis. Tissue excision was done under sterile conditions and properly labeled. All of the samples and excised tissues were preserved in 95% ethanol for future use. All procedures performed involving animals were approved by the ethical committee of the University of Karachi, approval no IBC KU-260/2022.

 

Amplification and sequencing of DNA barcode region

The DNA extraction was accomplished using a GeneJET DNA purification kit (Catalogue no. K0721). The recommended protocol for animal tissue DNA extraction was followed for DNA extraction. The product of DNA extraction was stored at -20°C for further experimental work.

The target region of 652 bp was amplified using published primers (Fish F1 and Fish R1) for DNA barcoding of fishes (Karim et al., 2016). PCR amplified product was sequenced commercially. Further, the excised tissues were also sent to the Canadian Centre for DNA barcoding (CCDB), the University of Guelph for sequencing and generation of DNA barcodes. Obtained sequences are uploaded to BOLDsystems for further analysis and also submitted to NCBI Genbank, the details are mentioned in Table 1.

Barcode index number allocation

Uploaded data on the BOLD workbench is automatically referred to a unique relevant BIN (Barcode Index Number). All the samples in one bin share the similarity at the molecular level. Available sequences of type species from across the globe were also downloaded and included in the analysis to explore genetic diversity. Species identification using molecular tools was also reconfirmed by BLAST search analysis. The significant E-values that are generated pairwise also play a paramount role in the delineation of fish species. If there was any mismatch between morphological identification and molecular identification then both were revisited to reach the concrete decision after the consideration of both parameters. The genetic distances based on the COI gene were determined with the help of MEGA X by using the Kimura 2 Parameter (K2P). Additionally, other analyses including a distance summary, nucleotide diversity, and a barcode gap analysis were done using the BOLD systems workbench, while the Fst analysis used R studio.

Phylogenetic relationship of experimental species from Pakistan with specimens across the globe

A phylogenetic analysis was conducted by using sequences obtained from experimental species from Pakistan and also retrieved data of the same species from across the globe. All of the sequences were aligned using the MSA/MAFT method. The trees to the explicit phylogenetic relationship were developed using the Fast tree method. Upon formation, the GGTree method was used for the visualization of relationships (Yu, 2020). The Genbank/Accession number for each species was manifested within the center of the tree. All of the work was done via UNIX, R, and R Studio. The species belonging to the same order and families were

 

Table 1: Geographical coordinates, sampling location, and GenBank accession number of rare species.

Family

Specie name

Order

Location

Voucher no.

Accession no

BOLD ID

Lactariidae

Lactarius lactarius

Perciformes

Latitude;

24°50'53.2"N

Longitude;

66°58'40.6"E

Fish Harbour Rd., West Wharf Karachi, Karachi City, Sindh, Pakistan

MAK-142

OQ801201

BOLD:AAD4634

Rachycentridae

Rachycentron canadum

Carangiformes

MAK-73

OQ807163

BOLD:AAB2939

Caesionidae

Caesio varilineata

Perciformes

MAK-103

OQ825963

BOLD:ADK7119

Pempheridae

Pempheris russellii

Perciformes

MAK-108

OQ826120

BOLD:AAD1777

Pomacanthidae

Pomacanthus annularis

Perciformes

MAK-59

OQ807215

BOLD:AAF1425

Holocentridae

Myripristis botche

Holocentri-formes

MAK-119

OQ807657

BOLD:AAX2837

Holocentridae

Sargocentron rubrum

Holocentri-formes

MAK-120

OQ808566

BOLD:AAB9306

Plotosidae

Plotosus lineatus

Siluriformes

MAK-37

OQ808970

BOLD:ABY8174

Chanidae

Chanos chanos

Gonoryn-chiformes

MAK-145

OQ809068

BOLD:AAC1320

Uranoscopidae

Uranoscopus dollfusi

Trachiniformes

MAK-114

OQ809069

BOLD:ACX9882

Terapontidae

Terapon jarbua

Perciformes

MAK-05

OQ810001

BOLD:AAA9351

Terapontidae

Terapon puta

Perciformes

MAK-13

OQ810033

BOLD:AAB0170

Drepaneidae

Drepane longimana

Perciformes

MAK-44

OQ814428

BOLD:AAB0170

scatophagidae

Scatophagus argus

Perciformes

MAK-92

OQ814532

BOLD:AAB3530

Stromateidae

Pampus argenteus

Scombriformes

MAK-27

OQ815713

BOLD:AAB6557

 

Table 2: Values obtained from barcode gap analysis.

Order

Family

Species

Max intra-Sp

Nearest species

Nearest neighbour

Distance to NN

Barcode gap

Carangiformes

Rachycentridae

Rachycentron canadum

2.74

Drepane longimana

SUFIS237-21

21.85

19.11

Holocentriformes

Holocentridae

Myripristis botche

0.74

Terapon jarbua

SUFIS385-21

17.17

16.43

Holocentriformes

Holocentridae

Sargocentron rubrum

4.23

Myripristis botche

SUFIS330-21

19.26

15.03

Moroniformes

Drepaneidae

Drepane longimana

0.31

Terapon jarbua

SUFIS005-20

19.26

18.95

Perciformes

Caesionidae

Caesio varilineata

0.96

Myripristis botche

SUFIS330-21

18.83

17.87

Perciformes

Pempheridae

Pempheris russellii

2.16

Sargocentron rubrum

SUFIS331-21

21.44

19.28

Perciformes

Pomacanthidae

Pomacanthus annularis

1.72

Myripristis botche

SUFIS329-21

22.02

20.3

Perciformes

Scatophagidae

Scatophagus argus

2.64

Pampus argenteus

SUFIS188-21

20.43

17.79

Perciformes

Terapontidae

Terapon jarbua

3.62

Myripristis botche

SUFIS329-21

17.17

13.55

Perciformes

Terapontidae

Terapon puta

1.59

Terapon jarbua

SUFIS386-21

19.63

18.04

Scombriformes

Stromateidae

Pampus argenteus

3.96

Caesio varilineata

SUFIS149-21

19.42

15.46

Siluriformes

Plotosidae

Plotosus lineatus

1.44

Scatophagus argus

SUFIS138-21

21.2

19.76

Trachiniformes

Uranoscopidae

Uranoscopus dollfusi

0.68

Sargocentron rubrum

SUFIS331-21

24.59

23.91

 

clustered under the same clade and the species belonging to different families and orders were clustered under different clades. So, a unique pattern of phylogeny was depicted based on COI nucleotide information.

Generation of DNA barcodes and QR codes

In the present era technology is very developed and Smartphones can widely be used to access 2D QR codes. At a single time, almost 4000 characters can be accessible, hence such technology can be used in fish identification coupled with genetic information. The sequence data were used to generate DNA barcodes and QR codes using online code generator tools (Ayesha et al., 2019).

Results and Discussion

A series of analyses were performed on sequence data obtained from the experimental species and data retrieved from online databases (NCBI, BOLDsystems). A total of 542 sequences including sequences from experimental species were considered for Phylogenetic and Fst analysis using R studio. The metadata of all the species is given in Supplementary Table 1.

Barcode gap analysis

The barcode gap can be measured by finding the distribution of intra-species distances and the distance between their nearest neighbors. The barcode gap can be an impressive tool to delineate the species boundaries via nucleotide sequence information. All of the data from experimental species were uploaded to the online database Bold systems and were tested to reveal the barcode gap between them using the bold systems workbench. Three scatterplots are provided to confirm the existence and magnitude of the Barcode Gap. The results are listed in Table 2. The first two scatterplots show the overlap of the max and mean intra-specific distances vs the inter-specific (nearest neighbor) distances. The third scatterplot plots the number of individuals in each species against their max intra-specific distances, as a test for sampling bias (Figure 2).

 

Genetic distances and nucleotide diversity

The genetic distances based on COI nucleotide data were measured by using the K2P model and Muscel alignment on the BOLD systems workbench. Distribution of distances on different levels was manifested between all specimens and showed 20.17, and 19.87 percentages within genus and family, respectively. Table 3 shows that thymine (T) is the most common nucleotide, with a mean percentage of 29.06%, followed by adenine (A) at 23.71%. The overall GC content is 47.23%, with the first codon position being the most GC-rich at 57.60%. Interestingly, the third codon position has a lot of variability, which could affect how genes are expressed and proteins are made.

 

Table 3: Summary statistics for nucleotide frequency distribution are provided in the table.

Nucleotide

Min

Mean

Max

SE

G %

17.04

18.42

20.22

0.0984

C %

24.88

28.81

33.57

0.2441

A %

20.22

23.71

25.35

0.1617

T %

25.08

29.06

32.57

0.2340

GC %

42.09

47.23

51.54

0.2923

GC % Codon Pos 1

55.30

57.60

59.72

0.1217

GC % Codon Pos 2

42.11

43.03

45.50

0.0821

GC % Codon Pos 3

27.19

41.05

53.93

0.8530

 

Fst analysis

Fraction of genetic variance manifests differentiation within a population. The sequence data from experimental species and already available sequence data from NCBI were taken for each species. Overall, 542 sequences representing 12 species were used in this analysis. The metadata of each sequence is provided in Supplementary Table 1. The results were illustrated by heatmap charts and principle component analysis (Figures 3, 4, 5, 6). Fst analysis and development of heatmaps and PCA charts was done by R script. Sequence data taken from NCBI represents different geographical regions of the world.

Chanos chanos

Experimental sequences of Chanos chanos and sequences downloaded from NCBI were considered for Fst to determine genetic differentiation between species populations. Fst analysis revealed a maximum genetic variance of 0.5 between populations of Australia/ Iran, China/ Iran, and Bangladesh/ Iran. Moreover, a minimum of 0.04 genetic variation was revealed within the species population of the USA and Bangladesh. The values for Fst were interpreted by using heatmap charts (Figure 3A) and PCA (Figure 4A). The countries having similar

 

 

divergence were interpreted under the same cluster. According to PCA interpretation of Fst, the maximum divergence was shown in the population of Bangladesh followed by France, the Philippines, and Pakistan.

Drepane longimana

The maximum genetic variance (0.48) was manifested by the study population of India and Bangladesh, while the minimum genetic variance of (0.09) was revealed between the species populations of Indonesia and South Africa (Figure 3B). The countries having related genetic divergence were clustered under the same cluster as compared to ones that have distant genetic variance. PCA from Fst values representing Drepane longimana manifested maximum genetic variation between the species population of Indonesia followed by South Africa and Pakistan (Figure 4B).

Lactarius lactarius

The COI-based Fst analysis of sequences of Lactarius lactarius shows a maximum value of 0.5 genetic variations from the study population of Taiwan and Pakistan. Moreover, the species population representing India and Pakistan showed the lowest genetic variance value which was 0.08. The results obtained from Fst were interpreted with heatmap charts (Figure 3C) and PCA (Figure 4C). The countries having less distant genetic variance within their population were seen to gather under a single cluster. Principle component analysis of the values obtained from Fst shows that the L. lactarius population of the Philippines shows significantly higher divergence followed by the population of China, Pakistan, and India.

 

Myripristis botche

From all sequences under study populations from multiple countries show the maximum genetic variance of 0.33. The minimum genetic variance was 0.1 as shown between populations of the Philippines and USA. The values of Fst were interpreted by a heatmap chart (Figure 3D) darker colors show greater values and lighter colors show lower values. Multiple countries illustrate the maximum genetic variance hence falling in darker regions. Moreover, species populations from Taiwan, Reunion, Pakistan, and South Africa made one cluster, and species populations from the USA and Philippines formed another cluster. PCA interpretation of Fst values showed a similar pattern (Figure 4D). The species population from South Africa showed maximum genetic variance followed by the population of Taiwan and Pakistan.

Pampus argenteus

Fst analysis showed maximum genetic variance (0.33) between study populations of Australia and the USA. Meanwhile, the study population from India and China showed a minimum Fst value of 0.05. PCA (Figure 4E) and heatmap charts (Figure 3E) were plotted by using values obtained from Fst. The darker colors on the heatmap represent high genetic variation and lighter colors on the heatmap chart display lower values. In heatmap charts countries that form a single cluster show relatively similar genetic variations among their populations like India, China, and Pakistan. PCA values indicate that Pakistan showed significantly higher divergence followed by Australia, China, and India.

Plotosus lineatus

The study populations of Plotosus lineatus representing different regions of the world were considered for Fst. Maximum genetic variance (0.75) was found between countries viz Taiwan/Pakistan, Taiwan/Australia, Reunion/Pakistan, Reunion/Australia, Japan/Taiwan, Indonesia/Taiwan, Lebanon/Reunion, and Taiwan/Australia. Similarly, minimum genetic divergence (0.05) was revealed between the species population of Lebanon and China based on COI as a potential marker. Heatmap (Figure 3F) and PCA (Figure 4D) were used for the illustration of values obtained from Fst data. Moreover, species populations from countries like Taiwan and Reunion form one cluster, meanwhile all other countries’ species population was clustered under separate clusters according to Fst values. PCA based on values obtained from Fst was also done and depicts that the Plotosus lineatus population of Pakistan is significantly more divergent followed by the population of the USA and Philippines.

Pomacanthus annularis

The species population of Pomacanthus annularis depicts a range of Fst values between 0.33-0.25 taken from different geographical regions of the world. Heatmap (Figure 5A) illustrated that the species population from Pakistan and Bangladesh formed one cluster meanwhile species populations representing different regions of the world were clustered under separate clusters concerning their Fst values. Principle component analysis (Figure 6A) depicts that the most significant divergence was produced by the population of Pakistan followed by Vietnam and India.

 

Rachycentron canadum

A maximum of 0.61 genetic divergence was revealed between Australia and the UK, meanwhile, a minimum of 0.06 genetic divergence was illustrated by species populations belonging to Mozambique and South Africa. So, the population species of Mozambique and South Africa are more related as compared to others. The heatmap chart depicts that species populations of countries having somehow similar genetic divergence were clustered under a single cluster, while species populations having different genetic variances were clustered under different clusters (Figure 5B). PCA analysis of data obtained from Fst showed that Bangladesh produced more significant divergence followed by Bangladesh, Pakistan, and the UK (Figure 6B).

Sargocentron rubrum

Study populations of this species belonging to different regions of the world were tested for genetic variance between them using Fst. It produces a maximum value of divergence of about 0.57 between the study population of Australia and Saudia Arabia, while minimum genetic variation (0.06) was revealed in the species populations of Australia and Indonesia. Countries that share somehow similar values were combined to form one cluster and countries that depict different colors were considered to have distant genetic divergence (Figure 5C). PCA analysis depicts that species population from Pakistan and the Philippines shows significantly higher differentiation followed by other countries (Figure 6C).

Scatophagus argus

Fst analysis revealed a maximum genetic variance (0.75) between the study population of Australia/Vietnam, and Pakistan/Vietnam, while a minimum genetic variance of 0.04 was found to be present in populations of India and Sri Lanka. The presence of low genetic variance between two populations indicates that both of them are related to each other. The study populations of countries that show similar genetic differentiation (Indonesia, China, Sri Lanka, India, and Malaysia) were clustered under the same cluster, on the other hand, species populations of (Vietnam) were clustered under separate clusters (Figure 5D). PCA analysis of values obtained from Fst illustrated that the study population of Australia showed a significantly high level of differentiation followed by that of Pakistan and China (Figure 6D).

Terapon jarbua

Terapon jarbua manifested a maximum of 0.56 genetic variance between the species population of Pakistan and Israel. While a minimum of 0.11 genetic variance was displayed by study populations of France and the Philippines. Figure 5E depicted heatmap chart of values obtained from the Fst Species population of (France/Philippines, Taiwan/ China/ India) having similar genetic differentiation formed a single cluster, and the population of (Australia, Mexico, and Israel) having different genetic variance formed different clusters. Principle component analysis (Figure 6E) showed significantly higher differentiation for the species populations from Pakistan followed by other countries.

Terapon puta

A maximum of 0.35 genetic variance was displayed between the species populations of Philippines/Pakistan, and Lebanon/Pakistan, while a minimum of 0.05 genetic variance was observed between populations of Pakistan and India hence they are more closely related to each other. A heatmap chart (Figure 5F) was used to manifest data obtained from Fst Species populations from India and Pakistan were clustered in a single cluster as they show similar genetic divergence and all other countries’ populations were gathered to form a separate cluster as they show different divergence levels. Fst values-based PCA (Figure 6F) was also conducted and showed that China shows a significantly higher level of differentiation followed by Lebanon and other countries.

Phylogenetic analysis among marine species occurring worldwide

Figure 7A illustrates a phylogenetic tree for species of Chanos chanos occurring worldwide to delineate evolutionary relationships and genetic similarities between them. Every country was highlighted with a unique pattern of color. The orange color represents sequences grabbed from NCBI and the blue color shows sequenced species from Pakistan. All species were clustered under clades according to evolutionary relationships. Sequences from Pakistan formed a clade surrounded by India and the Philippines. Figure 7B depicts the phylogenetic relationship of Drepane longimana each of the sequences representing different countries was clustered according to their evolutionary relationship. Type specimens from Pakistan formed a clade with species from Bangladesh and showed more similarity to sequences from Bangladesh and India. Figure 7C shows the evolutionary relationship of Lactarius lactarius occurring across the globe, the studied species from Pakistan were clustered under a clade with China, India, and USA. Figure 7D illustrates the phylogenetic tree of Myripristis botche. The experimental species were clustered under the clade representing species from Pakistan along with species from the island of Reunion. Figure 7E shows the phylogenetic relationship of Pampus argenteus samples occurring across the globe. The experimental samples from Pakistan were clustered together with sequences from India. Both regions of Pakistan share a common Arabian Sea so they also share common ancestors. Figure 7F illustrates the evolutionary relationship of Plotosus lineatus occurring across the globe. The experimental species formed a clade with the species from Saudi Arabia, India, and the Philippines. So, the individuals from these regions are more related to each other and are considered to have evolved from the same ancestors.

 

Figure 8A illustrates a phylogenetic relationship of Pomacanthus annularis members occurring in different regions of the world. The experimental species from Pakistan were clustered under a clade shared by species members from Myanmar and Sri Lanka. Figure 8B depicts the evolutionary relationship of worldwide occurring members belonging to the Rachycentron canadum species. Our experimental species were clustered under the clade in which the members from Bangladesh and India were clustered. So, the experimental species was evolutionary more similar to members from Bangladesh and India. Figure 8C illustrates the phylogenetic relationship between members of Sargocentron rubrum representing different regions of the world. The studied samples were clustered in a clade with members from India and Australia. Figure 8D represents the phylogenetic relationship of Scatophagus argus members from different regions of the world. All of the members were clustered under different clades according to their evolutionary relationship. Experimental species were clustered under a clade with Australia, Sri Lanka, and India. Figure 8E manifested phylogenetic relationship members of Terapon jarbua from available COI sequences on NCBI and sequences obtained from experimental samples. The blue color on the tips represents the type specimen and the orange color on the tips shows sequences obtained from NCBI. The studied species were clustered under the clade with the members of species from France and India. Figure 8F manifested the phylogenetic relationship of Terapon puta occurring across the globe. Each of the countries was labeled with a unique pattern of colors. The studied samples from Pakistan were clustered under a clade with members from Lebanon, Israel, and Saudi Arabia.

 

Phylogenetic relationship at the family level

Based on results obtained from phylogenetic analysis at the family level it was depicted that species belonging to the same family were clustered under the same clades whereas species belonging to different families were clustered under different clades (Figure 9). Moreover, the species members of Chanos chanos from Pakistan were more similar to South Africa, Qatar, and India. Species members of Myripristis botche formed a distinct clade and type member from Pakistan was surrounded by USA and Reunion depicting a close genetic resemblance. Plotosus lineatus from Pakistan showed close genetic resemblance with Saudia Arabia and Malaysia. Pampus argenteus members from Pakistan showed close resemblance with China and India. Species members of Scatophagus argus representing Pakistan were more closely related to India and Sri Lanka. Likewise, each of type species showed their resemblance with the members from countries that showed more closed evolutionary relationships or the members that were inferred from common ancestors.

 

The results based on mitochondrial gene COI show significant differentiation between the species population of (Lactarius lactarius, Rachycentron canadum, Caesio varilineata, Pempheris russellii, Pomacanthus annularis, Myripristis botche, Sargocentron rubrum, Plotosus lineatus, Chanos chanos, Uranoscopus dollfusi, Terapon jarbua, Terapon puta, Drepane longimana, Scatophagus argus, Pampus argenteus) belonging to 13 families and 7 orders, representing different regions of the world. To our knowledge, our study provides the first-ever COI nucleotide sequence addition of Caesio Varilineata, and Uranscopus dollfusi to Genbank.

This study revealed genetic differentiation between various fish populations via Fst analysis. The results showed a substantial genetic differentiation (Fst = 0.75) in populations of Plotosus lineatus and Scatophagus argus, illustrating a significant divergence between them. Other species displayed varying levels of genetic differentiation (Fst = 0.51, 0.57, 0.48, and 0.3), implying some degree of population divergence. Possible factors contributing to these differences can be due to geographical barriers, limited gene flow, and local adaptation. The findings have important implications for conservation efforts, as high levels of genetic differentiation may require tailored conservation strategies. Additionally, the study contributes to our understanding of the genetic structure of the studied fish species and underscores the importance of maintaining genetically diverse populations for species resilience and adaptability.

Within the suborder Stromateoidei the family Pempheridae is the most speciose family, this family illustrated the poorly resolved systematics owing to its highly conserved anatomical features and wide distribution (Haedrich, 1967; Liu et al., 2013). Previously work done on Pampus argenteus lacked comprehensive information regarding the phylogenetics and population divergence. They showed within species genetic distance of about 0.000 to 0.005, However, Fst analysis revealed 0.05-0.33 genetic differentiation within the population of the same species this variation can be due to the large sample size (Cui et al., 2010). Moreover, our study has illustrated the detailed phylogenetics and population differentiation.

Nasihin-Seth et al. (2019) provide the DNA barcodes of Plotosus lineatus based on COI and the limited phylogenetic information from Malaysia. However, the members of this species particularly from Pakistan were under-explored on a molecular basis. Our study manifested the detailed phylogenetic relation as well as the species’ genetic diversity and population divergence within different regions of the world. The divergence between the members of species belonging to different regions of the world was measured using Fst analysis and showed 0.05-0.75 genetic divergence. The species population of Plotosus lineatus from the Arabian Sea was more similar to the population of India as they share less genetic differentiation between them. This similarity is obvious, as Pakistan and India share a common border. Over time, species and their relatives may travel within these places and share common ancestors as well.

Barton et al. (2018) documented the detailed information of Rachycentron canadum on the taxonomic characters as well as based on COI sequencing of the members of species from the Australian Coast. However, the species data from Pakistan still need to be explored. Our study provides detailed information on basic morphology, DNA barcodes, population genetic divergence, and phylogenetic relationship between the complex diversity of the Arabian Sea and all of the COI sequence data available on the online databases. The population divergence was found using Fst analysis and shows (0.06-0.61) values. The type species population showed more resemblance to Chinese and Indian populations. The low population differentiation can be due to different factors, including geographical proximity, historical gene flow, shared environmental conditions, and high dispersal ability.

Washim et al. (2022) illustrated the information based on morphometric measurements of Scatophagus argus, particularly from Bangladesh. However, in addition to morphometric measurements molecular-based exploration is required for more authentic species delimitation, particularly from Pakistan. Our study offers comprehensive insights into DNA barcodes, population divergence, genetic distances, and phylogenetic relationships between the experimental species and other species found globally. The online QR code for the nucleotide information of sequences was also generated. The population divergence was revealed using Fst analysis and illustrated (0.04-0.75) values. The phylogenetic tree shows a grouping of the taxas based on nucleotide sequence similarity. The population differentiation of type species showed maximum resemblance with populations of India and China. The possible reason can be the geographical proximity as they share common borders and shorter distances for potential migration. There can be other reasons such as high dispersal ability, historical gene flow, and shared environmental conditions.

The Fst analysis based on a single gene, COI, unravels significant genetic differentiation among species populations of type species. The maximum value of 0.75 manifested considerable genetic divergence between different populations indicating limited gene flow and potential population isolation. In contrast minimal value of 0.04 implies a lower level of genetic divergence between other populations, depicting higher gene flow and potential connectivity. These findings depict that genetic structure within type species varies across regions and populations. Higher differentiation values proposed the presence of subpopulations or local adaptations, that are typically influenced by factors such as environmental conditions, geographical barriers, and historical events. The higher degree of gene flow is illustrated by the lower values, potentially facilitated by factors like geographical proximity, shared environmental conditions, or the active dispersal mechanism. The differentiation values obtained from a single gene, such as COI give an insight about genetic divergence at that particular locus. To get a detailed insight into genetic structure it is recommended to consider additional genetic markers and broad sampling across different genomic regions. The above findings highlighted the importance of maintaining connectivity between populations having low differentiation to preserve overall genetic diversity and to prevent the loss of locally adapted potential traits. In addition, populations that manifested higher differentiation may warrant specific conservation measures to protect their unique genetic resources and ensure their long-term viability. The present study could serve as an integrative genetic analysis that unravels differentiation and its implications for the conservation and management of type species populations.

Phylogenetic trees were constructed using R script delineating the evolutionary relationship between experimental species and species occurring across the globe. The species were clustered under the clades according to their orders, families, genera, and species. The pattern of clustering obtained from phylogenetic relationships unraveled an interesting genetic relationship among species occurring across the globe. The species members of Myripristis botche showed close resemblance with species members of Reunion. The island of Reunion is very far from the northern Arabian Sea but due to many natural changes and adaptations some species might have traveled and may share common ancestors at the time of course. Likewise, the other species revealed their evolutionary history and genetic similarity. These findings may manifest historical migration events or they may share common ancestors across the covered regions. Further studies may be required to reveal the true phylogeny of life for these groups of fishes and could serve as a valuable insight into evolutionary relationships, and practical implications for conservation, genetics, and ecology.

As a whole, the COI barcode data obtained from members of these 13 families have delineated species explicitly. However, only two species (Pempheris russellii and Caesio varilineata) were not able to be discriminated against and shared barcode index numbers with Pempheris nesogallica, Pempheris mangula, and Pterocaesio chrysozona respectively, keeping in mind their morphological characters were considered again and were matched with all members representing that bin, so a strong decision was made after considering morphological characters as we cannot rely solely on one method for identification. Moreover, there is a need for more molecular markers for the exact identification of these species.

Just like supermarkets that contain a specific QR code for each product, QR codes for species nucleotide sequences can easily be developed and accessed via mobile applications QR code scanners, etc. Before this study QR codes for fish species were developed (Ghouri et al., 2020) but codes for our experimental species were developed for the first time. Bio-Rad DNA barcodes are also being generated using online tools (Yang et al., 2019) for species identification on a molecular basis. Our study developed both DNA barcodes on BOLD systems and the QR codes containing the sequence information are given as a Supplementary Figure 1. As a whole DNA barcoding together with taxonomy can be an effective approach to developing strategies for the management, conservation, and monitoring of the fisheries sector. The present study targets some rare 15 species belonging to 13 families and 7 orders to explore them on morphology and molecular basis. DNA barcoding is not commonly practiced in Pakistan so identification on a molecular basis and generation of DNA-based QR codes can be validated as a basic approach for this purpose.

Conclusions and Recommendations

The present data could serve as a baseline for the identification of new species, environmental studies, and biogeographic patterns. It documented the COI-based DNA database of 15 rarely occurring marine species and two species (Caesio varilineata, Uranoscopus dollfusi) for the first time in Genbank from a coastal area inhabiting Pakistan. Moreover, the COI sequence of five species (Chanos chanos, Sargocentron rubrum, Plotosus lineatus, Myripristis botche, and Pomacanthus annularis) was first time documented from Pakistan to BOLD systems and NCBI repository. The QR codes and DNA barcodes for robust nucleotide information availability were introduced. Moreover, this study serves as a crucial tool for developing management and conservation strategies for marine fish diversity.

Declarations

Funding

We are delighted to accomplish the current research with the help of the Higher Education Commission of Pakistan (NRPU-10403). Sequencing work was performed at the Canadian Centre for DNA Barcoding (CCDB), University of Guelph, Canada.

IRB approval

This study was approved by the Institutional Review Board of University of Karachi approval no IBC KU-260/2022.

Ethical statement

This study utilized fish samples collected from local markets in Karachi, Pakistan, ensuring minimal harm and no direct impact on fish populations. No harmful or destructive sampling methods were employed.

Supplementary material

The supplementary material associated with this article is given after the references.

Conflict of interest

The authors have declared no conflict of interest.

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Supplementary Material

 

Supplementary Table 1: Metadata for each species included in the study.

Specie name

Accession no

Country

Type

Rachycentron canadum

OQ807163

Pakistan

Studied

Rachycentron canadum

OQ387350

Philippines

NCBI

Rachycentron canadum

OQ386365

Philippines

NCBI

Rachycentron canadum

OQ386340

Philippines

NCBI

Rachycentron canadum

OQ385628

Philippines

NCBI

Rachycentron canadum

OQ385483

Philippines

NCBI

Rachycentron canadum

KF809415

Philippines

NCBI

Rachycentron canadum

KJ202194

Philippines

NCBI

Table continued on next page..........

Specie name

Accession no

Country

Type

Rachycentron canadum

KF715002

Philippines

NCBI

Rachycentron canadum

KC970500

Philippines

NCBI

Rachycentron canadum

MT455681

USA

NCBI

Rachycentron canadum

KF461225

USA

NCBI

Rachycentron canadum

MN839528

USA

NCBI

Rachycentron canadum

MH378655

USA

NCBI

Rachycentron canadum

MH378586

USA

NCBI

Rachycentron canadum

HQ956531

Australia

NCBI

Rachycentron canadum

HQ956530

Australia

NCBI

Rachycentron canadum

EF609446

Australia

NCBI

Rachycentron canadum

KY372071

China

NCBI

Rachycentron canadum

KY372070

China

NCBI

Rachycentron canadum

KY372069

China

NCBI

Rachycentron canadum

KY372068

China

NCBI

Rachycentron canadum

KY372067

China

NCBI

Rachycentron canadum

EU600160

China

NCBI

Rachycentron canadum

EU600159

China

NCBI

Rachycentron canadum

EF607498

China

NCBI

Rachycentron canadum

KP266829

China

NCBI

Rachycentron canadum

JF494343

South Africa

NCBI

Rachycentron canadum

HQ945936

South Africa

NCBI

Rachycentron canadum

HQ945904

South Africa

NCBI

Rachycentron canadum

KF489739

South Africa

NCBI

Rachycentron canadum

KF489738

South Africa

NCBI

Rachycentron canadum

JF494342

Mozambique

NCBI

Rachycentron canadum

JF494341

Mozambique

NCBI

Rachycentron canadum

JQ841350

Belize

NCBI

Rachycentron canadum

JQ841349

Belize

NCBI

Rachycentron canadum

KU236035

Saudi Arabia

NCBI

Rachycentron canadum

MH235696

Myanmar

NCBI

Rachycentron canadum

MK777204

Taiwan

NCBI

Rachycentron canadum

MK777203

Taiwan

NCBI

Rachycentron canadum

KU943713

Taiwan

NCBI

Rachycentron canadum

KU943712

Taiwan

NCBI

Rachycentron canadum

KU943711

Taiwan

NCBI

Rachycentron canadum

MN458362

Bangladesh

NCBI

Rachycentron canadum

MN083099

Bangladesh

NCBI

Rachycentron canadum

MF588559

Bangladesh

NCBI

Rachycentron canadum

MF588558

Bangladesh

NCBI

Rachycentron canadum

MF588657

Bangladesh

NCBI

Rachycentron canadum

KU168730

UK

NCBI

Rachycentron canadum

KP410326

Pakistan

NCBI

Rachycentron canadum

KJ590063

India

NCBI

Rachycentron canadum

EF609586

India

NCBI

Rachycentron canadum

EF609585

India

NCBI

Rachycentron canadum

EF609584

India

NCBI

Table continued on next column..........

Specie name

Accession no

Country

Type

Rachycentron canadum

EF609583

India

NCBI

Rachycentron canadum

EF609582

India

NCBI

Rachycentron canadum

HQ589294

India

NCBI

Rachycentron canadum

HQ589293

India

NCBI

Rachycentron canadum

OL851817

India

NCBI

Lactarius lactarius

OQ801201

Pakistan

Studied

Lactarius lactarius

EF607416

China

NCBI

Lactarius lactarius

EF607417

China

NCBI

Lactarius lactarius

EF607418

China

NCBI

Lactarius lactarius

JQ681391

China

NCBI

Lactarius lactarius

MZ329509

China

NCBI

Lactarius lactarius

MZ329510

China

NCBI

Lactarius lactarius

MZ329511

China

NCBI

Lactarius lactarius

OL494216

China

NCBI

Lactarius lactarius

KY371638

China

NCBI

Lactarius lactarius

KY371639

China

NCBI

Lactarius lactarius

KY371640

China

NCBI

Lactarius lactarius

KY371641

China

NCBI

Lactarius lactarius

KY371642

China

NCBI

Lactarius lactarius

EF609529

India

NCBI

Lactarius lactarius

EF609530

India

NCBI

Lactarius lactarius

EF609531

India

NCBI

Lactarius lactarius

FJ347949

India

NCBI

Lactarius lactarius

MK775650

India

NCBI

Lactarius lactarius

MK775651

India

NCBI

Lactarius lactarius

MK775652

India

NCBI

Lactarius lactarius

MN747963

India

NCBI

Lactarius lactarius

MW167118

India

NCBI

Lactarius lactarius

MW167119

India

NCBI

Lactarius lactarius

HQ564485

Indonesia

NCBI

Lactarius lactarius

HQ564486

Indonesia

NCBI

Lactarius lactarius

JN312892

Indonesia

NCBI

Lactarius lactarius

MH085843

Indonesia

NCBI

Lactarius lactarius

KT883634

USA

NCBI

Lactarius lactarius

KT883657

USA

NCBI

Lactarius lactarius

KU535572

Philippines

NCBI

Lactarius lactarius

KU943707

Taiwan

NCBI

Lactarius lactarius

MK777483

Taiwan

NCBI

Lactarius lactarius

MH429314

Bangladesh

NCBI

Lactarius lactarius

MK340629

Bangladesh

NCBI

Lactarius lactarius

MN511922

Pakistan

NCBI

Pomacanthus annularis

OQ807215

Pakistan

Studied

Pomacanthus annularis

MH235689

Myanmar

NCBI

Pomacanthus annularis

MK340680

Bangladesh

NCBI

Pomacanthus annularis

MK340679

Bangladesh

NCBI

Pomacanthus annularis

MH085785

Indonesia

NCBI

Table continued on next page..........

Specie name

Accession no

Country

Type

Pomacanthus annularis

KU944248

Taiwan

NCBI

Pomacanthus annularis

KF268138

India

NCBI

Pomacanthus annularis

KF268137

India

NCBI

Pomacanthus annularis

KF268136

India

NCBI

Pomacanthus annularis

FJ583876

Sri Lanka

NCBI

Pomacanthus annularis

FJ583875

Vietnam

NCBI

Myripristis botche

OQ807657

Pakistan

Studied

Myripristis botche

KU943300

Taiwan

NCBI

Myripristis botche

JX390744

USA

NCBI

Myripristis botche

OQ385782

USA

NCBI

Myripristis botche

OQ387325

Philippines

NCBI

Myripristis botche

MF409583

Reunion

NCBI

Myripristis botche

KF489654

South Africa

NCBI

Myripristis botche

MK777368

Vietnam

NCBI

Sargocentron rubrum

OQ808566

Pakistan

Studied

Sargocentron rubrum

OQ387824

Philippines

NCBI

Sargocentron rubrum

OQ387696

Philippines

NCBI

Sargocentron rubrum

OQ386629

Philippines

NCBI

Sargocentron rubrum

KJ202198

Philippines

NCBI

Sargocentron rubrum

MZ421439

Vietnam

NCBI

Sargocentron rubrum

MK777374

Vietnam

NCBI

Sargocentron rubrum

MK777373

Vietnam

NCBI

Sargocentron rubrum

MK777372

Vietnam

NCBI

Sargocentron rubrum

MK777371

Vietnam

NCBI

Sargocentron rubrum

MK777370

Vietnam

NCBI

Sargocentron rubrum

GU673963

Indonesia

NCBI

Sargocentron rubrum

GU673717

Indonesia

NCBI

Sargocentron rubrum

MN870501

Indonesia

NCBI

Sargocentron rubrum

MN870474

Indonesia

NCBI

Sargocentron rubrum

MN870057

Indonesia

NCBI

Sargocentron rubrum

MN869979

Indonesia

NCBI

Sargocentron rubrum

MH049175

Indonesia

NCBI

Sargocentron rubrum

MH190810

Indonesia

NCBI

Sargocentron rubrum

GU673204

Australia

NCBI

Sargocentron rubrum

HQ956403

Australia

NCBI

Sargocentron rubrum

HQ956402

Australia

NCBI

Sargocentron rubrum

KR861551

Lebanon

NCBI

Sargocentron rubrum

KC501252

Turkey

NCBI

Sargocentron rubrum

KC501251

Turkey

NCBI

Sargocentron rubrum

KC501250

Turkey

NCBI

Sargocentron rubrum

KC501249

Turkey

NCBI

Sargocentron rubrum

KC501248

Turkey

NCBI

Sargocentron rubrum

KC501247

Turkey

NCBI

Sargocentron rubrum

KC501246

Turkey

NCBI

Sargocentron rubrum

KC501245

Turkey

NCBI

Sargocentron rubrum

KC501244

Turkey

NCBI

Table continued on next column..........

Specie name

Accession no

Country

Type

Sargocentron rubrum

KC501243

Turkey

NCBI

Sargocentron rubrum

KC501242

Turkey

NCBI

Sargocentron rubrum

KC501241

Turkey

NCBI

Sargocentron rubrum

KC501240

Turkey

NCBI

Sargocentron rubrum

KC501239

Turkey

NCBI

Sargocentron rubrum

KC501238

Turkey

NCBI

Sargocentron rubrum

KC501237

Turkey

NCBI

Sargocentron rubrum

KC501236

Turkey

NCBI

Sargocentron rubrum

KC501235

Turkey

NCBI

Sargocentron rubrum

KC501234

Turkey

NCBI

Sargocentron rubrum

KC501233

Turkey

NCBI

Sargocentron rubrum

KY176608

Turkey

NCBI

Sargocentron rubrum

JQ623980

Turkey

NCBI

Sargocentron rubrum

EU600150

China

NCBI

Sargocentron rubrum

EU600149

China

NCBI

Sargocentron rubrum

FJ237913

China

NCBI

Sargocentron rubrum

FJ237912

China

NCBI

Sargocentron rubrum

FJ265867

India

NCBI

Sargocentron rubrum

MK962512

India

NCBI

Sargocentron rubrum

KU987435

India

NCBI

Sargocentron rubrum

KJ632830

India

NCBI

Sargocentron rubrum

KJ632826

India

NCBI

Sargocentron rubrum

KJ466132

India

NCBI

Sargocentron rubrum

KJ466131

India

NCBI

Sargocentron rubrum

KF442242

India

NCBI

Sargocentron rubrum

MK340699

Bangladesh

NCBI

Sargocentron rubrum

MK340698

Bangladesh

NCBI

Sargocentron rubrum

MK340697

Bangladesh

NCBI

Sargocentron rubrum

MH331859

Saudi Arabia

NCBI

Sargocentron rubrum

KU943292

Taiwan

NCBI

Plotosus lineatus

OQ808970

Pakistan

Studied

Plotosus lineatus

EF607323

China

NCBI

Plotosus lineatus

EF607324

China

NCBI

Plotosus lineatus

EU595234

China

NCBI

Plotosus lineatus

EU595235

China

NCBI

Plotosus lineatus

EU595236

China

NCBI

Plotosus lineatus

EU595237

China

NCBI

Plotosus lineatus

EU595238

China

NCBI

Plotosus lineatus

EU595239

China

NCBI

Plotosus lineatus

FJ238012

China

NCBI

Plotosus lineatus

MG220589

China

NCBI

Plotosus lineatus

MK264717

China

NCBI

Plotosus lineatus

EU148553

India

NCBI

Plotosus lineatus

FJ237535

India

NCBI

Plotosus lineatus

FJ384696

India

NCBI

Plotosus lineatus

KF268171

India

NCBI

Table continued on next page..........

Specie name

Accession no

Country

Type

Plotosus lineatus

KF268172

India

NCBI

Plotosus lineatus

KF268173

India

NCBI

Plotosus lineatus

KF268174

India

NCBI

Plotosus lineatus

KF824841

India

NCBI

Plotosus lineatus

KF824842

India

NCBI

Plotosus lineatus

KF824843

India

NCBI

Plotosus lineatus

KP296155

India

NCBI

Plotosus lineatus

EU490870

USA

NCBI

Plotosus lineatus

FJ918911

USA

NCBI

Plotosus lineatus

FJ583870

Philippines

NCBI

Plotosus lineatus

FJ583871

Philippines

NCBI

Plotosus lineatus

FJ583872

Philippines

NCBI

Plotosus lineatus

FJ583873

Philippines

NCBI

Plotosus lineatus

FJ583874

Philippines

NCBI

Plotosus lineatus

KF604684

Philippines

NCBI

Plotosus lineatus

KF604685

Philippines

NCBI

Plotosus lineatus

KF604686

Philippines

NCBI

Plotosus lineatus

KF604687

Philippines

NCBI

Plotosus lineatus

KF604688

Philippines

NCBI

Plotosus lineatus

KF604689

Philippines

NCBI

Plotosus lineatus

KF604690

Philippines

NCBI

Plotosus lineatus

KF809412

Philippines

NCBI

Plotosus lineatus

HQ956387

Australia

NCBI

Plotosus lineatus

JF494186

South Africa

NCBI

Plotosus lineatus

JF494187

South Africa

NCBI

Plotosus lineatus

JF494188

South Africa

NCBI

Plotosus lineatus

JF494189

South Africa

NCBI

Plotosus lineatus

JF952819

Japan

NCBI

Plotosus lineatus

JN313096

Indonesia

NCBI

Plotosus lineatus

JQ350233

Reunion

NCBI

Plotosus lineatus

KM538482

Israel

NCBI

Plotosus lineatus

KM538483

Israel

NCBI

Plotosus lineatus

KM538484

Israel

NCBI

Plotosus lineatus

KM538485

Israel

NCBI

Plotosus lineatus

KM538486

Israel

NCBI

Plotosus lineatus

KM538487

Israel

NCBI

Plotosus lineatus

KM538488

Israel

NCBI

Plotosus lineatus

KM538489

Israel

NCBI

Plotosus lineatus

KM538490

Israel

NCBI

Plotosus lineatus

KM538491

Israel

NCBI

Plotosus lineatus

KM538492

Israel

NCBI

Plotosus lineatus

KM538493

Israel

NCBI

Plotosus lineatus

KM538494

Israel

NCBI

Plotosus lineatus

KM538495

Israel

NCBI

Plotosus lineatus

KM538496

Israel

NCBI

Plotosus lineatus

KM538497

Israel

NCBI

Table continued on next column..........

Specie name

Accession no

Country

Type

Plotosus lineatus

KM538498

Israel

NCBI

Plotosus lineatus

KM538499

Israel

NCBI

Plotosus lineatus

KM538500

Israel

NCBI

Plotosus lineatus

KM538501

Israel

NCBI

Plotosus lineatus

KM538502

Israel

NCBI

Plotosus lineatus

KM538503

Israel

NCBI

Plotosus lineatus

KM538504

Israel

NCBI

Plotosus lineatus

KP221606

Malaysia

NCBI

Plotosus lineatus

KP221607

Malaysia

NCBI

Plotosus lineatus

KP221608

Malaysia

NCBI

Plotosus lineatus

KP258657

Malaysia

NCBI

Plotosus lineatus

KP258658

Malaysia

NCBI

Plotosus lineatus

KP258659

Malaysia

NCBI

Plotosus lineatus

KR861548

Lebanon

NCBI

Plotosus lineatus

KU179077

Saudi Arabia

NCBI

Plotosus lineatus

KU499687

Saudi Arabia

NCBI

Plotosus lineatus

MH331830

Saudi Arabia

NCBI

Plotosus lineatus

KU943007

Taiwan

NCBI

Chanos chanos

OQ809068

Pakistan

Studied

Chanos chanos

DQ884995

South Africa

NCBI

Chanos chanos

DQ884996

South Africa

NCBI

Chanos chanos

DQ885083

Australia

NCBI

Chanos chanos

DQ885085

Australia

NCBI

Chanos chanos

KJ669401

Australia

NCBI

Chanos chanos

EU752071

USA

NCBI

Chanos chanos

EU752072

USA

NCBI

Chanos chanos

KF929723

USA

NCBI

Chanos chanos

GU674244

Indonesia

NCBI

Chanos chanos

KP856764

Indonesia

NCBI

Chanos chanos

KP856765

Indonesia

NCBI

Chanos chanos

KP856766

Indonesia

NCBI

Chanos chanos

KU692424

Indonesia

NCBI

Chanos chanos

KU692425

Indonesia

NCBI

Chanos chanos

KU692426

Indonesia

NCBI

Chanos chanos

HQ149824

Iran

NCBI

Chanos chanos

HQ654696

Philippines

NCBI

Chanos chanos

HQ654697

Philippines

NCBI

Chanos chanos

HQ654698

Philippines

NCBI

Chanos chanos

HQ654699

Philippines

NCBI

Chanos chanos

HQ654700

Philippines

NCBI

Chanos chanos

OQ385882

Philippines

NCBI

Chanos chanos

OQ386950

Philippines

NCBI

Chanos chanos

OQ387320

Philippines

NCBI

Chanos chanos

JN242677

China

NCBI

Chanos chanos

JN242678

China

NCBI

Chanos chanos

JN242679

China

NCBI

Table continued on next column..........

Specie name

Accession no

Country

Type

Chanos chanos

JQ431600

France

NCBI

Chanos chanos

JQ431601

France

NCBI

Chanos chanos

KP308089

India

NCBI

Chanos chanos

LT669927

India

NCBI

Chanos chanos

MK301234

India

NCBI

Chanos chanos

MK301235

India

NCBI

Chanos chanos

MK902719

India

NCBI

Chanos chanos

DQ885084

Taiwan

NCBI

Chanos chanos

KU893046

Taiwan

NCBI

Chanos chanos

KU942899

Taiwan

NCBI

Chanos chanos

KU942900

Taiwan

NCBI

Chanos chanos

KU942901

Taiwan

NCBI

Chanos chanos

KU942902

Taiwan

NCBI

Chanos chanos

KY802063

UK

NCBI

Chanos chanos

KY802076

UK

NCBI

Chanos chanos

MK216571

Qatar

NCBI

Chanos chanos

MK216572

Qatar

NCBI

Chanos chanos

MK216573

Qatar

NCBI

Chanos chanos

MK216574

Qatar

NCBI

Chanos chanos

MK216575

Qatar

NCBI

Chanos chanos

MK216576

Qatar

NCBI

Chanos chanos

MK216577

Qatar

NCBI

Chanos chanos

MK216578

Qatar

NCBI

Chanos chanos

MK241873

Qatar

NCBI

Chanos chanos

MK241874

Vietnam

NCBI

Chanos chanos

MK241875

Vietnam

NCBI

Chanos chanos

MK241876

Vietnam

NCBI

Chanos chanos

MK241877

Vietnam

NCBI

Chanos chanos

MK241878

Vietnam

NCBI

Chanos chanos

MK241879

Vietnam

NCBI

Chanos chanos

MK241880

Vietnam

NCBI

Chanos chanos

MK241881

Vietnam

NCBI

Chanos chanos

MK241882

Vietnam

NCBI

Chanos chanos

MK241883

Vietnam

NCBI

Chanos chanos

MK241884

Vietnam

NCBI

Chanos chanos

MK777366

Vietnam

NCBI

Chanos chanos

MN083123

Bangladesh

NCBI

Terapon jarbua

OQ810001

Pakistan

Studied

Terapon jarbua

EF607573

China

NCBI

Terapon jarbua

EF607574

China

NCBI

Terapon jarbua

EF607575

China

NCBI

Terapon jarbua

EF607576

China

NCBI

Terapon jarbua

EF607577

China

NCBI

Terapon jarbua

EF607578

China

NCBI

Terapon jarbua

EF607579

China

NCBI

Terapon jarbua

EF607580

China

NCBI

Table continued on next column..........

Specie name

Accession no

Country

Type

Terapon jarbua

EU871691

China

NCBI

Terapon jarbua

EU871692

China

NCBI

Terapon jarbua

FJ237549

India

NCBI

Terapon jarbua

FJ265859

India

NCBI

Terapon jarbua

FJ347885

India

NCBI

Terapon jarbua

FJ347886

India

NCBI

Terapon jarbua

FJ347887

India

NCBI

Terapon jarbua

FJ384681

India

NCBI

Terapon jarbua

JX260979

India

NCBI

Terapon jarbua

JX983498

India

NCBI

Terapon jarbua

KC241987

India

NCBI

Terapon jarbua

KC417308

India

NCBI

Terapon jarbua

KC774674

India

NCBI

Terapon jarbua

KF268188

India

NCBI

Terapon jarbua

KF268189

India

NCBI

Terapon jarbua

KJ920134

India

NCBI

Terapon jarbua

KM079294

India

NCBI

Terapon jarbua

KM079295

India

NCBI

Terapon jarbua

HQ149959

Iran

NCBI

Terapon jarbua

HQ149960

Iran

NCBI

Terapon jarbua

HQ149961

Iran

NCBI

Terapon jarbua

JF494663

South Africa

NCBI

Terapon jarbua

JF494664

South Africa

NCBI

Terapon jarbua

JF494665

South Africa

NCBI

Terapon jarbua

JF494666

South Africa

NCBI

Terapon jarbua

JN021254

Philippines

NCBI

Terapon jarbua

JN021255

Philippines

NCBI

Terapon jarbua

KC970423

Philippines

NCBI

Terapon jarbua

KF009671

Philippines

NCBI

Terapon jarbua

KF715030

Philippines

NCBI

Terapon jarbua

KF715031

Philippines

NCBI

Terapon jarbua

KJ202209

Philippines

NCBI

Terapon jarbua

KJ202210

Philippines

NCBI

Terapon jarbua

JQ342095

France

NCBI

Terapon jarbua

JQ342096

France

NCBI

Terapon jarbua

JQ342097

France

NCBI

Terapon jarbua

JQ342098

France

NCBI

Terapon jarbua

JQ342099

France

NCBI

Terapon jarbua

JQ342100

France

NCBI

Terapon jarbua

JQ342101

France

NCBI

Terapon jarbua

JQ342102

France

NCBI

Terapon jarbua

JQ342103

France

NCBI

Terapon jarbua

JQ342104

France

NCBI

Terapon jarbua

JQ342105

France

NCBI

Terapon jarbua

JQ342106

France

NCBI

Terapon jarbua

JQ342107

France

NCBI

Table continued on next column..........

Specie name

Accession no

Country

Type

Terapon jarbua

JQ342108

France

NCBI

Terapon jarbua

JQ342109

France

NCBI

Terapon jarbua

JQ342110

France

NCBI

Terapon jarbua

JQ342111

France

NCBI

Terapon jarbua

JQ342112

France

NCBI

Terapon jarbua

JQ741340

Mexico

NCBI

Terapon jarbua

KJ466137

Israel

NCBI

Terapon jarbua

KJ466138

Israel

NCBI

Terapon jarbua

KP194256

Australia

NCBI

Terapon jarbua

KP194261

Australia

NCBI

Terapon jarbua

KP194552

Australia

NCBI

Terapon jarbua

KP194928

Australia

NCBI

Terapon jarbua

KP204162

Taiwan

NCBI

Terapon jarbua

KP204163

Taiwan

NCBI

Terapon jarbua

KP204164

Taiwan

NCBI

Terapon jarbua

KP204165

Taiwan

NCBI

Terapon jarbua

KP204166

Taiwan

NCBI

Terapon jarbua

KP204167

Taiwan

NCBI

Terapon jarbua

KP204168

Taiwan

NCBI

Terapon jarbua

KP204169

Taiwan

NCBI

Terapon jarbua

KP204170

Taiwan

NCBI

Terapon jarbua

KP204171

Taiwan

NCBI

Terapon jarbua

KP204172

Taiwan

NCBI

Terapon jarbua

KP204173

Taiwan

NCBI

Terapon jarbua

KP204174

Taiwan

NCBI

Terapon jarbua

KP204175

Taiwan

NCBI

Terapon jarbua

KP204176

Taiwan

NCBI

Terapon jarbua

KP204177

Taiwan

NCBI

Terapon jarbua

KP204178

Taiwan

NCBI

Terapon jarbua

KP204179

Taiwan

NCBI

Terapon jarbua

KP204180

Taiwan

NCBI

Terapon jarbua

KP204181

Taiwan

NCBI

Terapon jarbua

KP204182

Taiwan

NCBI

Terapon jarbua

KP204183

Taiwan

NCBI

Terapon jarbua

KP204184

Taiwan

NCBI

Terapon jarbua

KP204185

Taiwan

NCBI

Terapon jarbua

KP204186

Taiwan

NCBI

Terapon jarbua

KP204187

Taiwan

NCBI

Terapon jarbua

KP204188

Taiwan

NCBI

Terapon jarbua

KP204189

Taiwan

NCBI

Terapon jarbua

KP204190

Taiwan

NCBI

Terapon jarbua

KP204191

Taiwan

NCBI

Terapon jarbua

KP204192

Taiwan

NCBI

Terapon jarbua

KP204193

Taiwan

NCBI

Terapon puta

OQ810033

Pakistan

Studied

Terapon puta

KC774675

India

NCBI

Table continued on next column..........

Specie name

Accession no

Country

Type

Terapon puta

KC774676

India

NCBI

Terapon puta

KJ920126

India

NCBI

Terapon puta

KP308091

India

NCBI

Terapon puta

KP308092

India

NCBI

Terapon puta

KX064469

India

NCBI

Terapon puta

KX064470

India

NCBI

Terapon puta

KX064471

India

NCBI

Terapon puta

MK348200

India

NCBI

Terapon puta

MK348201

India

NCBI

Terapon puta

MK902721

India

NCBI

Terapon puta

KF564317

Israel

NCBI

Terapon puta

KF809425

Philippines

NCBI

Terapon puta

KR861565

Lebanon

NCBI

Terapon puta

KU499747

Saudi Arabia

NCBI

Terapon puta

KU499748

Saudi Arabia

NCBI

Terapon puta

KU499749

Saudi Arabia

NCBI

Terapon puta

KU499750

Saudi Arabia

NCBI

Terapon puta

KU499751

Saudi Arabia

NCBI

Terapon puta

KY372206

China

NCBI

Terapon puta

KY372207

China

NCBI

Terapon puta

KY372208

China

NCBI

Terapon puta

KY372209

China

NCBI

Terapon puta

MN512110

Pakistan

NCBI

Drepane longimana

OQ814428

Pakistan

Studied

Drepane longimana

FJ459579

India

NCBI

Drepane longimana

GU674215

Indonesia

NCBI

Drepane longimana

GU674218

Indonesia

NCBI

Drepane longimana

GU674220

Indonesia

NCBI

Drepane longimana

JF493392

South Africa

NCBI

Drepane longimana

JF493393

South Africa

NCBI

Drepane longimana

JF493394

South Africa

NCBI

Drepane longimana

JF493395

South Africa

NCBI

Drepane longimana

MH429335

Bangladesh

NCBI

Drepane longimana

MK340610

Bangladesh

NCBI

Drepane longimana

MK340611

Bangladesh

NCBI

Drepane longimana

MT012667

Bangladesh

NCBI

Drepane longimana

MN511883

Pakistan

NCBI

Drepane longimana

MN511884

Pakistan

NCBI

Scatophagus argus

OQ814532

Pakistan

Studied

Scatophagus argus

DQ107757

Australia

NCBI

Scatophagus argus

EF607516

China

NCBI

Scatophagus argus

EF607517

China

NCBI

Scatophagus argus

EF607518

China

NCBI

Scatophagus argus

EF607519

China

NCBI

Scatophagus argus

KP260476

China

NCBI

Scatophagus argus

KT951732

China

NCBI

Table continued on next column..........

Specie name

Accession no

Country

Type

Scatophagus argus

KX254482

China

NCBI

Scatophagus argus

KX254483

China

NCBI

Scatophagus argus

KY372108

China

NCBI

Scatophagus argus

KY372109

China

NCBI

Scatophagus argus

KY372110

China

NCBI

Scatophagus argus

EF609604

India

NCBI

Scatophagus argus

EF609605

India

NCBI

Scatophagus argus

EF609606

India

NCBI

Scatophagus argus

EF609607

India

NCBI

Scatophagus argus

FJ347948

India

NCBI

Scatophagus argus

FJ584086

India

NCBI

Scatophagus argus

JX983493

India

NCBI

Scatophagus argus

KC774668

India

NCBI

Scatophagus argus

KJ920130

India

NCBI

Scatophagus argus

KM079335

India

NCBI

Scatophagus argus

KM079336

India

NCBI

Scatophagus argus

KP212376

India

NCBI

Scatophagus argus

KP212377

India

NCBI

Scatophagus argus

KP296156

India

NCBI

Scatophagus argus

KY009860

India

NCBI

Scatophagus argus

KY634864

India

NCBI

Scatophagus argus

KY634866

India

NCBI

Scatophagus argus

MG923401

India

NCBI

Scatophagus argus

MG923402

India

NCBI

Scatophagus argus

MG923403

India

NCBI

Scatophagus argus

MG923404

India

NCBI

Scatophagus argus

MG923405

India

NCBI

Scatophagus argus

FJ584087

Sri Lanka

NCBI

Scatophagus argus

FJ584088

Sri Lanka

NCBI

Scatophagus argus

FJ584089

Sri Lanka

NCBI

Scatophagus argus

FJ584090

Sri Lanka

NCBI

Scatophagus argus

GU674034

Indonesia

NCBI

Scatophagus argus

GU674036

Indonesia

NCBI

Scatophagus argus

KU692847

Indonesia

NCBI

Scatophagus argus

KU692848

Indonesia

NCBI

Scatophagus argus

KU692849

Indonesia

NCBI

Scatophagus argus

KU692850

Indonesia

NCBI

Scatophagus argus

MG921203

Indonesia

NCBI

Scatophagus argus

MG921207

Indonesia

NCBI

Scatophagus argus

MH085813

Indonesia

NCBI

Scatophagus argus

MH085814

Indonesia

NCBI

Scatophagus argus

JN021246

Philippines

NCBI

Scatophagus argus

JN021247

Philippines

NCBI

Scatophagus argus

JN021250

Philippines

NCBI

Scatophagus argus

KF715006

Philippines

NCBI

Scatophagus argus

KF715007

Philippines

NCBI

Table continued on next column..........

Specie name

Accession no

Country

Type

Scatophagus argus

KF715008

Philippines

NCBI

Scatophagus argus

KF809418

Philippines

NCBI

Scatophagus argus

KF930378

Thailand

NCBI

Scatophagus argus

KF930379

Thailand

NCBI

Scatophagus argus

KU944950

Taiwan

NCBI

Scatophagus argus

KU944951

Taiwan

NCBI

Scatophagus argus

KU944952

Taiwan

NCBI

Scatophagus argus

KX223946

Malaysia

NCBI

Scatophagus argus

KX223947

Malaysia

NCBI

Scatophagus argus

KX223948

Malaysia

NCBI

Scatophagus argus

KX281938

Malaysia

NCBI

Scatophagus argus

MH674067

Malaysia

NCBI

Scatophagus argus

MH721188

Vietnam

NCBI

Pampus argenteus

OQ815713

Pakistan

Studied

Pampus argenteus

DQ107596

Australia

NCBI

Pampus argenteus

DQ107597

Australia

NCBI

Pampus argenteus

DQ107598

Australia

NCBI

Table continued on next column..........

Specie name

Accession no

Country

Type

Pampus argenteus

DQ107599

Australia

NCBI

Pampus argenteus

DQ107600

Australia

NCBI

Pampus argenteus

EF607457

China

NCBI

Pampus argenteus

EF607458

China

NCBI

Pampus argenteus

EF607459

China

NCBI

Pampus argenteus

EF607460

China

NCBI

Pampus argenteus

EU119289

China

NCBI

Pampus argenteus

EU119292

China

NCBI

Pampus argenteus

EU119293

China

NCBI

Pampus argenteus

EU595224

China

NCBI

Pampus argenteus

HM068249

China

NCBI

Pampus argenteus

EU752147

USA

NCBI

Pampus argenteus

EU752148

USA

NCBI

Pampus argenteus

FJ226531

India

NCBI

Pampus argenteus

FJ226532

India

NCBI

Pampus argenteus

FJ226533

India

NCBI

Pampus argenteus

FJ384702

India

NCBI

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Pakistan Journal of Zoology

October

Pakistan J. Zool., Vol. 56, Iss. 5, pp. 2001-2500

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